Techniques for production of useful proteins using genetic engineering are widely used nowadays. Among these, expression systems using Escherichia coli as a host are the most commonly used expression systems. Many proteins have been produced using recombinants. A so-called expression vector is generally constructed and used for the production of useful proteins using recombinants. In the expression vector, a gene of interest is placed under the control of a promoter which is recognized by an RNA polymerase. Exemplary promoters used for expression vectors for Escherichia coli as a host are lac, trp, tac, gal and ara promoters. Expression vectors that utilize promoters other than those directly recognized by Escherichia coli RNA polymerase include the pET-system (Novagen). The pET-system utilizes a promoter recognized by an RNA polymerase from bacteriophage T7 which infects Escherichia coli (see J. Mol. Biol., 189:113–130 (1986); Gene, 56:125–135 (1987)). In case of the pET-system, T7 RNA polymerase is expressed in Escherichia coli, a gene of interest placed downstream of T7 promoter in an expression vector is transcribed by T7 RNA polymerase, and the protein of interest is synthesized using the translation system of the host.
However, if a protein of interest is expressed at a high level using one of many Escherichia coli expression systems including the pET-system, the protein of interest may form an insoluble complex called inclusion body in many cases. As a result, the amount of the protein of interest in its active form is greatly reduced. It has been reported for several polypeptides that active polypeptides were obtain by solubilization and refolding of inclusion bodies. The recovery rates are generally low in many cases. In addition, appropriate refolding conditions need to be examined for each protein of interest. Thus, a system for directly expressing an active protein in Escherichia coli has been desired.
It is considered that inclusion bodies are formed as a result of the following. An intermediate of a translated polypeptide chain prior to folding into its proper conformation is interwound with another polypeptide chain due to intermolecular interaction to form a huge insoluble complex. In such a case, it is known that the expression level of a protein in its active form is increased by culturing recombinant Escherichia coli cells at a temperature lower than the conventional one 37° C. (20 to 30° C.). It is supposed that this is because the slow translation by ribosome provides a sufficient time for the intermediate to be folded into its proper structure, and the slow action of intracellular proteolytic enzyme under the low-temperature conditions increases the stability of expressed active protein. Thus, attention has been paid to a method in which recombinant Escherichia coli cells are cultured under low-temperature conditions as being useful for producing a protein that forms inclusion bodies.
If a culture temperature for Escherichia coli cells during the logarithmic growth phase is lowered from 37° C. to 10–20° C., growth of the Escherichia coli cells is temporarily arrested, during which expression of a group of proteins called cold shock proteins is induced. The proteins are classified based on the induction level into two groups: a group I (10-fold or more) and a group II (less than 10-fold). Proteins in the group I include CspA, CspB, CspG and CsdA (see J. Bacteriol., 178:4919–4925 (1996); J. Bacteriol., 178:2994–2997 (1996)). Since the expression level of CspA (WO 90/09447) reaches 13% of the total cellular protein 1.5 hours after temperature shift from 37° C. to 10° C. (see Proc. Natl. Acad. Sci. USA, 87:283–287 (1990)), attempts have been made to utilize the promoter for the cspA gene for production of a recombinant protein at a low temperature.
Regarding a system for expressing a recombinant protein under low-temperature conditions using the cspA gene, the following effectiveness has been shown in addition to the above-mentioned highly efficient transcription initiation by the promoter for the gene at a low temperature.
(1) If mRNA that is transcribed from the cspA gene and capable of being translated does not encode a functional CspA protein (specifically, if it encodes only a portion of the N-terminal sequence of the CspA protein), such mRNA inhibits expression of other Escherichia coli proteins including cold shock proteins for a long period of time. During this period, the mRNA is preferentially translated (J. Bacteriol., 178:4919–4925 (1996); WO 98/27220). This phenomenon is called LACE (low temperature-dependent antibiotic effect of truncated cspA expression) effect.
(2) A sequence consisting of 15 nucleotides called a downstream box is located 12 nucleotides downstream of the initiation codon of the cspA gene. The translation efficiency under low-temperature conditions is made high due to this sequence.
(3) A 5′-untranslated region consisting of 159 nucleotides is located between the transcription initiation site and the initiation codon in the mRNA for the cspA gene. This region has a negative effect on the expression of CspA at 37° C. and a positive effect under low-temperature conditions.
In particular, the phenomenon as described in (1) above suggests the feasibility of specific expression of only a protein of interest utilizing the cspA gene. Thus, it is expected that the system can be applied to production of highly pure recombinant proteins or preparation of isotope-labeled proteins for structural analyses.
It is known that it may be difficult to culture an Escherichia coli cell containing an expression vector to a level at which the cell can be subjected to induction, or even construction of an expression vector may be impossible if expression control of the promoter for the gene is incomplete and the gene product is harmful to the host (see, for example, U.S. Pat. No. 5,654,169).
Modification of the expression vector has been tried using the 5′-untranslated region of the cspA gene in order to solve the above-mentioned problems, to further increase the gene expression efficiency, and to readily obtain the expressed product (WO 99/27117). Modification by introducing an operator for making the expression control strict or by introducing a mutation into the 5′-untranslated region for increasing the gene expression level is disclosed therein.